Induction of beta cell differentiation in human cells

ABSTRACT

The present invention provides methods for inducing insulin gene expression in cultured pancreas cells, the method comprising contacting a culture of endocrine pancreas cells expressing a PDX-1 gene and a NeuroD/BETA2 gene with a GLP-1 receptor agonist, wherein the cells have been cultured under conditions such that the cells are in contact with other cells in the culture, thereby inducing insulin gene expression in the cells. The invention also provides high throughput screening methods for modulators of β-cell function, stable cultures of cells made by the methods of the invention, and methods of treating a human subject using the methods of the invention.

CROSS REFERENCE OF RELATED PATENT APPLICSTIONS

[0001] The present application is related to U.S. patent applicationSer. No. (USSN) 09/522,376, filed Mar. 10, 2000, which is explicitlyincorporated herein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant No. 5R01 DKK55283-02 and 5 R01 DK55065-02, awarded by the National Institutesof Health. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Transplantation of cells exhibiting glucose-responsive insulinsecretion has the potential to cure diabetes. However, this approach islimited by an inadequate supply of cells with that property, with isexhibited only by pancreatic β-cells. The development of expandedpopulations of human β-cells that can be used for cell transplantationis therefore a major goal of diabetes research (D. R. W. Group,“Conquering diabetes: a strategic plan for the 21st century” NIHPublication No. 99-4398 (National Institutes of Health, 1999)). A numberof alternative approaches are being pursued to achieve that goal,including using porcine tissue as a xenograft (Groth et al., J Mol Med77:153-4 (1999)), expansion of primary human β-cells with growth factorsand extracellular matrix (Beattie et al., Diabetes 48:1013-9 (1999)),and generation of immortalized cell lines that exhibitglucose-responsive insulin secretion (Levine, Diabetes/MetabolismReviews 1: 209-46 (1997)).

[0004] Although there has been great interest in using porcine islets,they are difficult to manipulate in vitro and concerns have been raisedabout endogenous and exogenous xenobiotic viruses being transmitted tograft recipients (Weiss, Nature 391:327-8 (1998)). With primary humanβ-cells, entry into the cell cycle can be achieved using hepatocytegrowth factor/scatter factor (“HGF/SF”) plus extracellular matrix(“ECM”) (Beattie et al., Diabetes 48:1013-9 (1999), Hayek et al.,Diabetes 44:1458-1460 (1995)). However, this combination, whileresulting in a 2-3×10⁴-fold expansion in the number of cells, is limitedby cellular senescence and loss of differentiated function, particularlypancreatic hormone expression (Beattie et al., Diabetes 48:1013-9(1999)).

[0005] Immortalized cell lines from the human endocrine pancreas havebeen created to develop β-cell lines that exhibit glucose responsiveinsulin secretion (Wang et al., Cell Transplantation 6:59-67 (1997),Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen etal., Molecular and Cellular Biology 19:1864-1870 (1999)). The cell linesare made by infecting primary cultures of cells from various sourcesincluding adult islets, fetal islets, and purified β-cells, with viralvectors expressing the potent dominant oncogenes such as SV40 T antigenand H-ras^(val12) (Wang et al., Cell Transplantation 6:59-67 (1997),Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen etal., Molecular and Cellular Biology 19:1864-1870 (1999); see also U.S.Pat. No. 5,723,333). The combined effect of those oncogenes is totrigger growth factor-independent and extracellular matrix(ECM)-independent entry into the cell cycle, as well as to prolong thelifespan of the cells from 10-15 population doublings or primary cellsto approximately 150 doubling for the oncogene-expressing cells(Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)).Further introduction of the gene encoding the hTRT component oftelomerase results in immortalization, allowing the cells to be grownindefinitely (Halvorsen et al., Molecular and Cellular Biology19:1864-1870 (1999)).

[0006] Although the cell lines grow indefinitely, they losedifferentiated function, similar to growth-stimulated primary β-cells.Methods of stimulating differentiation of the cell lines intoinsulin-secreting β-cells are therefore desired. Such cells could thenbe transplanted in vivo as a treatment for diabetes.

SUMMARY OF THE INVENTION

[0007] Induction of β-cell differentiation in cultured human β-cells wasachieved by stimulating multiple signaling pathways, including thosedownstream of the homeodomain transcription factors NeuroD/BETA2 andPDX-1, cell-cell contact, and the glucagon-like peptide-1 (GLP-1)receptor. Synergistic activation of those pathways resulted indifferentiation of the cultured human β-cells, which initially expressno detectable pancreatic hormones, into fully functional β-cells thatexhibit glucose-responsive insulin secretion. Furthermore, these cellscan be transplanted in vivo and demonstrate glucose-responsiveexpression of insulin. The ability to grow unlimited quantities offunctional human β-cells in vitro provides the means for a definitivecell transplantation therapy for treatment of diabetes.

[0008] The expression of the transcription factor NeuroD/BETA2 in humanβ-cells that express PDX-1, are in cell to cell contact, and arecontacted with a GLP-1 receptor agonist resulted in certain desirablecharacteristics. For example, surprisingly, the resulting cells producehigh levels of insulin compared to cells that do not expressNeuroD/BETA2. Moreover, the cells expressing NeuroD/BETA2 are highlystable in cell culture and can be grown in culture for multiplegenerations. Thus, in some embodiments of the invention, the presentinvention provides a method for inducing insulin gene expression incultured endocrine pancreas cells, the method comprising the steps of(i) expressing a recombinant NeuroD/BETA2 polynucleotide and arecombinant PDX-1 gene in cells that have been cultured under conditionssuch that the cells are in contact with other cells in the culture; and(ii) contacting the cells with a GLP-1 receptor agonist, therebyinducing insulin gene expression in the cells. In some embodiments, thecells do not initially produce any detectable pancreatic hormones suchas insulin and glucagon.

[0009] In another aspect, the present invention provides a method ofidentifying a compound that modulates β-cell function, the methodcomprising the steps of contacting cells made by the method describedabove with the compound and determining the effect of the compound onβ-cell function.

[0010] In another aspect, the present invention provides a stableculture of endocrine pancreas cells, wherein the cells are in contactwith other cells in the culture, wherein the cells express a recombinantNeuroD/BETA2 gene and a recombinant PDX-1 gene, and wherein insulin geneexpression is stimulated in the cells when exposed to an effectiveamount of a GLP-1 receptor agonist.

[0011] In another aspect, the present invention provides a method oftreating a diabetic subject by providing to the subject cells thatsecrete insulin in response to glucose, the method comprising the stepsof: (i) contacting a culture of endocrine pancreas cells expressing aNeuroD/BETA2 gene and a PDX-1 gene with a GLP-1 receptor agonist,wherein the cells have been cultured under conditions such that thecells are in contact with other cells in the culture; and (ii)administering the cells to the subject, thereby providing to the subjectcells that secrete insulin in response to glucose.

[0012] In some embodiments, the GLP-1 receptor agonist is a GLP-1 analogor has an amino acid sequence of a naturally occurring peptide. In someembodiments, the GLP-1 receptor agonist is GLP-1, exendin-3, orexendin-4.

[0013] In some embodiments, the cells are cultured as aggregates insuspension.

[0014] In one embodiment, the cells are human cells. In anotherembodiment, the cells are βlox5 cells. A deposit of the βlox5 cells,which are human pancreatic cells, was made on Jul. 19, 2001 underaccession number PTA-3532 at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209.

[0015] In some embodiments, the cells express a recombinant oncogene. Insome embodiments, the cells express a recombinant oncogene. In someembodiments, the cells express a recombinant oncogene. In someembodiments, the cells express a recombinant telomerase gene.

[0016] In some embodiments, the diabetic subject is a human. In someembodiments, the subject has Type I insulin dependent diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1. Flow cytometric analysis of FAD autofluorescence in asingle cell suspension of human islets used to develop the βlox5 cellline.

[0018]FIG. 2: RT-PCR analysis of pancreatic hormone gene expression inβlox5. The conditions tested were βlox5 cells grown in monolayerculture, βlox5 cells infected with a retroviral vector expressing PDX-1,βlox5 cells grown as three-dimensional aggregates, and βlox5 cellstreated with exendin-4. FIG. 2(A) insulin; FIG. 2(B) quantitative RT-PCRanalysis of insulin gene expression; FIG. 2(C) other pancreatichormones. Exendin-4 (Sigma) was used at a concentration of 10 nM. RT-PCRfor insulin, somatostatin, glucagon, and IAPP have been describedpreviously (Itkin-Ansari et al., submitted). Quantitative RT-PCR wasdone by interpolation from a standard curve constructed using a plasmidcontaining the human insulin cDNA.

[0019]FIG. 3: Analysis of transcription factors expressed in βlox5cells. FIG. 3(A) electrophoretic mobility shift assay (EMSA) of PDX-1.EMSA for PDX-1 was performed using a probe derived from the humaninsulin promoter A5 element. FIGS. 3(B&C) RT-PCR analysis of BETA2 andPax6. FIG. 3(D) Western blot analysis of CREB. FIG. 3(E) EMSA of RIPE3b.

[0020]FIG. 4. Insulin protein in induced βlox5 cells. FIG. 4(A) Insulinimmunohistochemistry; FIG. 4(B) Insulin western blot analysis ofconditioned medium.

[0021]FIG. 5. Analysis of glucose-responsive insulin secretion. FIG.5(A) RT-PCR for glucokinase. FIG. 5(B) Radioimmunoassay for insulinsecreted from induced βlox5 cells grown in culture medium containingincreasing concentrations of glucose. Induced βlox5 cells were culturedin DME containing a single concentration of added glucose for one hour.Medium was harvested and assayed for insulin by RIA.

[0022]FIG. 6. RIA of c-peptide in nude mice transplanted with inducedβlox5 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Introduction

[0024] Using a cell line that initially exhibited few β-cellcharacteristics, despite having been originally derived from humanβ-cells, complete β-cell function has been successfully induced invitro. In addition, these cells have been transplanted into mice and areshown to produce insulin in a glucose responsive manner. Induction ofβ-cell differentiation in cells such as βlox5 cells requires threeinducing factors, PDX-1, cell-cell contact, and GLP-1 receptor agonistssuch as exendin-4. Moreover, expression of the transcription factor,NeuroD/BETA2, in combination with expression of PDX-1, contact with aGLP-1 agonist, and cell-to-cell contact in β-cell lines surprisinglyresults in greatly increased insulin production. Furthermore, β-cellsexpressing NeuroD/BETA2 in combination with expression of PDX-1, contactwith a GLP-1 agonist, and cell-to-cell contact, are also highly stablecell cultures that can replicate through many generations. Thus, theinvention provides methods of creating cells that produce a high levelof insulin over many generations.

[0025] Cultured cell lines such as TRM-6 (δ-cell lineage) and βlox5(β-cell lineage) are powerful tools that should allow identification ofthe full complement of genes that are required for endocrine celldevelopment and function. Furthermore, the requirement for multipleinteracting inducing factors provides an opportunity to study howdifferent signal transduction pathways interact with one another tocontrol a complex differentiation program.

[0026] The availability of an unlimited source of functional humanβ-cells has important implications for diabetes. One straightforwardapplication is in exploring aspects of β-cell biology that would benefitfrom an unlimited, homogeneous source of cells. High-throughputscreening for new diabetes drugs is one such application. The cells ofthe invention can be used, e.g., to screen for small molecule ormacromolecule GLP-1 receptor agonists or other compounds that enhanceinsulin expression. In addition, cells such as those described hereincan be used in a cell transplantation therapy for diabetes. Cells thatexpress PDX-1 and are in cell-to-cell contact with other cells arestimulated with a GLP-1 receptor agonist, as described herein. Suchcells are then transplanted into a suitable mammalian host, preferably ahuman. Such cells exhibit glucose-responsive insulin secretion in vivo.

[0027] In one embodiment, cultured cells useful in the practice of theinvention express one or more oncogenes, such as SV40 T antigen andHras^(val12), which minimally transform the cells but stimulate growthand bypass cellular senescence. Other suitable oncogenes include, e.g.,HPV E7, HPV E6, c-myc, and CDK4 (see also U.S. Pat. No. 5,723,333). Inaddition, the cells can be transduced with an oncogene encodingmammalian telomerase, such as hTRT, to facilitate immortalization.Suitable oncogenes can be identified by those of skill in the art, andpartial lists of oncogenes are provided in Bishop et al., RNA TumorViruses, vol. 1, pp. 1004-1005 (Weiss et al., eds, 1984), and Watson etal., Molecular Biology of the Gene (4^(th) ed. 1987). In some cases theoncogenes provide growth factor-independent and ECM-independent entryinto the cell cycle. Often the oncogenes are dominant oncogenes. Thecells can be analyzed for recombinant oncogene expression by analysis ofoncogene RNA or protein expression. Integration of an oncogene into thegenome can be confirmed, e.g., by Southern blot analysis. Often, theoncogenes are delivered to the cells using a viral vector, preferably aretroviral vector, although any suitable expression vector can be usedto transduce the cells (see, e.g., U.S. Pat. No. 5,723,333, whichdescribes construction of vectors encoding one or more oncogenes andtransduction of pancreas endocrine cells, see also Halvorsen et al.,Molecular and Cellular Biology 19:1864-1870 (1999)).

[0028] The vector used to create the cell lines incorporates recombinasesites, such as lox sites, so that the oncogenes can be deleted byexpression of a recombinase, such as the cre recombinase, in the cellsfollowing expansion (Halvorsen et al., Molecular and Cellular Biology19:1864-1870 (1999)). Deletion of the oncogenes is useful for cells thatare to be transplanted in to a mammalian subject. Other recombinasesystems include Saccharomyces cerevisiae FLP/FRT, lambda att/Int, Rrecombinase of Zygosaccharomyces rouxii. In addition, transposableelements and transposases could be used. Deletion of the oncogene can beconfirmed, e.g., by analysis of oncogene RNA or protein expression, orby Southern blot analysis.

[0029] The cultured cells of the invention express either endogenous orrecombinant NeuroD/BETA2 having NeuroD/BETA2 activity, e.g., alleles,polymorphic variants, and orthologs (see, e.g., U.S. Pat. No. 5,795,723;Miyachi, T., et al. Mol. Brain Res. 69, 223-231 (1999); Lee, et al.Science 268:836-844 (1995); Wilson et al., Nature 368, 32-38 (1994);Naya et al., Genes Dev. 9:1009-1019 (1995)). Human NeuroD/BETA2 allelesand variants are particularly desirable. Recombinant PDX-1 is deliveredto the cells using expression vectors, e.g., viral vectors such asretroviral vectors, as described above.

[0030] The cultured cells of the invention also express eitherendogenous or recombinant PDX-1 having PDX-1 activity, e.g., alleles,polymorphic variants, and orthologs (see, e.g., Sander et al., J. Mol.Med. 71:327-340 (1997)). Endogenous expression of PDX-1 can be inducedusing transcription factors such as hepatocyte nuclear factor 3 beta,which is involved in pancreatic β-cell expression of the PDX-1 gene(see, e.g., Wu et al, Molecular and Cellular Biology 17:6002-6013(1997)). Recombinant PDX-1 is delivered to the cells using expressionvectors, e.g., viral vectors such as retroviral vectors, as describedabove.

[0031] The vectors used to transduce the cells can be any suitablevector, including viral vectors such as retroviral vectors. Preferably,the vector is one that provides stable transformation of the cells, asopposed to transient transformation.

[0032] This invention relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994)).

[0033] GLP-1 receptor agonists, which are administered to the cells ofthe invention, include naturally occurring peptides such as GLP-1,exendin-3, and exendin-4 (see, e.g., U.S. Pat. No. 5,424,286; U.S. Pat.No. 5,705,483, U.S. Pat. No. 5,977,071; U.S. Pat. No. 5,670,360; U.S.Pat. No. 5,614,492), GLP-1 analogs (see, e.g., U.S. Pat. No. 5,545,618and U.S. Pat. No. 5,981,488), and small molecule analogs. GLP-1 receptoragonists may be tested for activity as described in U.S. Pat. No.5,981,488. Cells are contacted with a GLP-1 receptor agonist in a timeand amount effective to induce insulin mRNA expression, as describedherein. Typically, the cells are contacted with the GLP-1 receptoragonists for a discrete time period, as the GLP-1 receptor agonist isbelieved to act as a switch for insulin gene expression. Continuousadministration of the GLP-1 receptor agonist is therefore not required.

[0034] Without intending to bind the invention to a particular theory ofaction, it is believed that cell-to-cell contact may result in Myc downregulation. As demonstrated herein, down regulation of c-Myc inpancreatic cells is associated with differentiation of cells andproduction of insulin. Therefore, methods of antagonizing Myc are usefulto induce differentiation of cells. For example, PDX-1 expression,contact with a GLP-1 agonist and contact or expression of a Mycantagonist can be used to stimulate insulin expression in pancreaticcells. In some embodiments, the cells are also cultured in aggregates orsimilar structures to provide for maximal cell-to-cell contact. In someembodiments, NeuroD/BETA2 is also expressed in the cells.

[0035] Exemplary Myc antagonists include antisense molecules or nucleicacid catalysts (e.g., ribozymes) that target Myc transcripts. Expressioncassettes containing promoters (e.g., constitutive or inducible) can beoperably linked to polynucleotides coding for antisense RNA. Expressionof such constructs results in decreased translation of the c-Myc geneproduct. In another example, polypeptides that antagonize Myc function,such as MAD1 (Cultraro, et al. Curr. Topics Micro. Immunol. 224: 149-158(1997)), can be expressed in cells. In a third example, small moleculeswhich antagonize c-myc are contacted to pancreatic cells.

[0036] Immune rejection of grafted cells has previously been a majorobstacle to successful islet transplantation. Any universal human donorcell will be recognized by the immune system as an allograft. However,recent advances in therapy for allograft rejection may make this less ofa concern (see, e.g., Kenyon et al., Proc. Natl Acad. Sci. USA 96:8132-7(1999)). An advantage of using an immortalized cell line as a source oftransplantable cells is that they can be engineered to exhibit desirablequalities, including avoidance or suppression of host immune responses.

[0037] Cell Culture

[0038] This invention relies upon routine techniques in the field ofcell culture, and suitable methods can be determined by those of skillin the art using known methodology (see, e.g., Freshney et al., Cultureof Animal Cells (3^(rd) ed. 1994)). In general, the cell cultureenvironment includes consideration of such factors as the substrate forcell growth, cell density and cell contract, the gas phase, the medium,and temperature.

[0039] The cells of the invention are grown under conditions thatprovide for maximal cell to cell contact. For instance, in someembodiments, the cell-to-cell contact occurs to a greater degree thanfound in monolayer cell cultures. In a preferred embodiment, the cellsare grown in suspension as three dimensional aggregates. Suspensioncultures can be achieved by using, e.g., a flask with a magnetic stirreror a large surface area paddle, or on a plate that has been coated toprevent the cells from adhering to the bottom of the dish. In apreferred embodiment, the cells are grown in Costar dishes that havebeen coated with a hydrogel to prevent them from adhering to the bottomof the dish.

[0040] For the cells of the invention that are cultured under adherentconditions, plastic dishes, flasks, roller bottles, or microcarriers insuspension are used. Other artificial substrates can be used such asglass and metals. The substrate is often treated by etching, or bycoating with substances such as collagen, chondronectin, fibronectin,and laminin. The type of culture vessel depends on the cultureconditions, e.g., multi-well plates, petri dishes, tissue culture tubes,flasks, roller bottles, and the like.

[0041] Cells are grown at optimal densities that are determinedempirically based on the cell type. For example, a typical cell densityfor βlox5 cultures varies from 1×10³ to 1×10⁷ cells per ml. Cells arepassaged when the cell density is above optimal.

[0042] Cultured cells are normally grown in an incubator that provides asuitable temperature, e.g., the body temperature of the animal fromwhich is the cells were obtained, accounting for regional variations intemperature. Generally, 37° C. is the preferred temperature for cellculture. Most incubators are humidified to approximately atmosphericconditions.

[0043] Important constituents of the gas phase are oxygen and carbondioxide. Typically, atmospheric oxygen tensions are used for cellcultures. Culture vessels are usually vented into the incubatoratmosphere to allow gas exchange by using gas permeable caps or bypreventing sealing of the culture vessels. Carbon dioxide plays a rolein pH stabilization, along with buffer in the cell media and istypically present at a concentration of 1-10% in the incubator. Thepreferred CO₂ concentration typically is 5%.

[0044] Defined cell media are available as packaged, premixed powders orpresterilized solutions. Examples of commonly used media include DME,RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium (see, e.g.,GibcoBRL/Life Technologies Catalogue and Reference Guide; SigmaCatalogue). Typically, low glucose DME or RPMI 1640 are used in themethods of the invention. Defined cell culture media are oftensupplemented with 5-20% serum, typically heat inactivated, e.g., humanhorse, calf, and fetal bovine serum. Typically, 10% fetal bovine serumis used in the methods of the invention. The culture medium is usuallybuffered to maintain the cells at a pH preferably from 7.2-7.4. Othersupplements to the media include, e.g., antibiotics, amino acids,sugars, and growth factors such as hepatocyte growth factor/scatterfactor.

[0045] Expression of PDX-1 alone in a cell can result in arrested cellgrowth, thereby preventing cell division and production of a continuousculture of insulin producing cells. However, co-expression withNeuroD/BETA2 reverses the arrested cell growth phenotype and allows forcontinuous culture (i.e., with multiple cell divisions) ofinsulin-producing cells. In some embodiments, the pancreatic cellsdivide at least twice and preferably at least about 5 or at least about10 times while producing insulin or retaining the ability to produceinsulin.

[0046] Pharmaceutical Compositions and Administration

[0047] Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered (e.g., a cell or smallmolecule), as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed., 1989).

[0048] Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. In the practice of thisinvention, compositions can be administered, for example, by directsurgical transplantation under the kidney, intraportal administration,intravenous infusion, or intraperitoneal infusion.

[0049] Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets. The dose administered to a patient, inthe context of the present invention should be sufficient to effect abeneficial therapeutic response in the patient over time. The dose willbe determined by the efficacy of the particular cells employed and thecondition of the patient, as well as the body weight or surface area ofthe patient to be treated. The size of the dose also will be determinedby the existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, or transduced celltype in a particular patient.

[0050] In determining the effective amount of the cells to beadministered in the treatment or prophylaxis of conditions owing todiminished or aberrant insulin expression, the physician evaluates celltoxicity, transplantation reactions, progression of the disease, and theproduction of anti-cell antibodies. For administration, cells of thepresent invention can be administered in an amount effective to providenormalized glucose responsive-insulin production and normalized glucoselevels to the subject, taking into account the side-effects of the celltype at various concentrations, as applied to the mass and overallhealth of the patient. Administration can be accomplished via single ordivided doses.

[0051] Assays for Modulators of B-Cell Function

[0052] A. Assays

[0053] Assays using the cultured cells of the invention can be used totest for inhibitors and activators of β-cell function, e.g., insulinproduction and/or glucose responsive insulin production. Such modulatorsare useful for treating various disorders involving glucose metabolism,such as diabetes and hypoglycemia. Treatment of dysfunctions include,e.g., diabetes mellitus (all types); hyperinsulinism caused byinsulinoma, drug-related, e.g., sulfonylureas or excessive insulin,immune disease with insulin or insulin receptor antibodies, etc. (see,e.g., Harrison 's Internal Medicine (14^(th) ed. 1998)).

[0054] Modulation is tested using the cultures of the invention bymeasuring insulin gene expression, optionally with administration ofglucose, e.g., analysis of insulin mRNA expression using northern blot,dot blot, PCR, oligonucleotide arrays, and the like; and analysis ofinsulin protein expression (preproinsulin, proinsulin, insulin, orc-peptide) using, e.g., western blots, radio immune assays, ELISAs, andthe like. Downstream effects of insulin modulation can also be examined.Physical or chemical changes can be measured to determine the functionaleffect of the compound on β cell function. Samples or assays that aretreated with a potential inhibitor or activator are compared to controlsamples without the test compound, to examine the extent of modulation.

[0055] B. Modulators

[0056] The compounds tested as modulators of β-cell function can be anysmall chemical compound, or a macromolecule, such as a protein, sugar,nucleic acid or lipid. Typically, test compounds will be small chemicalmolecules and peptides. Essentially any chemical compound can be used asa potential modulator or ligand in the assays of the invention, althoughmost often compounds can be dissolved in aqueous or organic (especiallyDMSO-based) solutions are used. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays). It will be appreciated that there are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and the like.

[0057] In one preferred embodiment, high throughput screening methodsinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (potential modulatoror ligand compounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

[0058] A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

[0059] Preparation and screening of combinatorial chemical libraries iswell known to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT PublicationNo. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomerssuch as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. Nos. 5,506,337; benzodiazepines, 5,288,514, and thelike).

[0060] Devices for the preparation of combinatorial libraries arecommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0061] The assays can be solid phase or solution phase assays. In thehigh throughput assays of the invention, it is possible to screen up toseveral thousand different modulators or ligands in a single day. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 96 modulators. If 1536 well plates are used, thena single plate can easily assay from about 100-about 1500 differentcompounds. It is possible to assay many plates per day; assay screensfor up to about 6,000, 20,000, 50,000, or 100,000 or more differentcompounds is possible using the integrated systems of the invention.

[0062] Definitions

[0063] As used herein, the following terms have the meanings ascribed tothem unless specified otherwise.

[0064] “Inducing insulin gene expression” refers to increasing, in acell or culture of cells, the level of expression from the insulin geneby at least about 10% or more, preferably 25%, 50%, 100%, 500%, 1000%,5000%, or higher, as compared to a negative control culture. Insulingene expression can be measured by methods known to those of skill inthe art, e.g., by measuring insulin RNA expression, preproinsulin,proinsulin, insulin, or c-peptide production, e.g., using PCR,hybridization, and immunoassays.

[0065] Cells that “secrete insulin in response to glucose” are cells ora cell culture that, in comparison to a negative control (eithernon-insulin responsive cells or insulin responsive cells that are notexposed to glucose), have increased insulin secretion in response toglucose of at least about 10%, preferably 25%, 50%, 100%, 500%, 1000%,5000%, or higher than the control cells (measured as described above).

[0066] “Culturing cells” so that the cells are “in contact with othercells in the culture” refers to culture conditions that allow cell tocell contact. Under such conditions, many but not all cells are incontact with one or more other cells of the culture. Such conditionsinclude culturing the cells on a solid surface, such as a plate or abead, or culturing the cells in suspension such that the cell-to-cellcontact is greater than in cells grown in monolayer culture. Examples ofsuch conditions include growth of cells in three-dimensional aggregates.

[0067] “Endocrine pancreas cells” refers to cells originally derivedfrom an adult or fetal pancreas, preferably islet cells. “Cultured”endocrine pancreas cells refers to primary cultures as well as cellsthat have been transformed with genes such as an oncogene, e.g., SV40 Tantigen, ras, or a telomerase gene (e.g., hTRT).

[0068] A “GLP-1 receptor agonist” refers to GLP-1, a GLP-1 analog, or anaturally occurring peptide that binds to the GLP-1 receptor (e.g.,exendin-3 or -4), thereby activating signal transduction from thereceptor.

[0069] “Culturing” refers to growing cells ex vivo or in vitro. Culturedcells can be non-naturally occurring cells, e.g., cells that have beentransduced with an exogenous gene such as an oncogene or a transcriptionfactor such as NeuroD/BETA2 and/or PDX-1. Cultured cells can also benaturally occurring isolates or primary cultures.

[0070] “Pancreatic hormones” refer to hormones synthesized by thepancreas and include, e.g., insulin and glucagon.

[0071] A “stable” cell line or culture is one that can grow in vitro foran extended period of time, such as for at least about 50 celldivisions, or for about 6 months, more preferably for at least about 150cell divisions, or at least about ten months, and more preferably atleast about a year.

[0072] “Modulating β-cell function” refers to a compound that increases(activates) or decreases (inhibits) glucose responsive insulin secretionof an endocrine pancreas cell. Glucose responsive insulin secretion canbe measured by a number of methods, including analysis of insulin mRNAexpression, preproinsulin production, proinsulin production, insulinproduction, and c-peptide production, using standard methods known to ofskill in the art. To examine the extent of modulation, cultured cellsare treated with a potential activator or inhibitor and are compared tocontrol samples without the activator or inhibitor. Control samples(untreated with inhibitors or activators) are assigned a relativeinsulin value of 100%. Inhibition is achieved when the insulin valuerelative to the control is about 90%, preferably 75%, 50%, and morepreferably 25-0%. Activation is achieved when the insulin value relativeto the control is 110%, more preferably 125%, 150%, and most preferablyat least 200-500% higher or 1000% or higher.

[0073] “Transduction” refers to any method of delivering an exogenousnucleic acid, e.g., an expression vector, to a cell, includingtransfection, lipofection, electroporation, viral transduction,microinjection, particle bombardment, receptor mediated endocytosis, andthe like.

[0074] A diabetic subject is a mammalian subject, often a human subject,that has any type of diabetes, including primary and secondary diabetes,type 1 NIDDM-transient, type 1 IDDM, type 2 IDDM-transient, type 2NIDDM, and type 2 MODY, as described in Harrison 's Internal Medicine,14th ed. 1998.

[0075] “Expressing” a gene refers to expression of a recombinant orendogenous gene, e.g., resulting in mRNA or protein production from thegene. A recombinant gene can be integrated into the genome or in anextrachromosomal element.

[0076] “Antibody” refers to a polypeptide comprising a framework regionfrom an immunoglobulin gene or fragments thereof that specifically bindsand recognizes an antigen. The recognized immunoglobulin genes includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0077] An exemplary immunoglobulin (antibody) structural unit comprisesa tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

[0078] Antibodies exist, e.g., as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

[0079] For preparation of monoclonal or polyclonal antibodies, anytechnique known in the art can be used (see, e.g., Kohler & Milstein,Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc. (1985)). Techniques for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produceantibodies to polypeptides of this invention. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanizedantibodies. Alternatively, phage display technology can be used toidentify antibodies and heteromeric Fab fragments that specifically bindto selected antigens (see, e.g., McCafferty et al., Nature 348:552-554(1990); Marks et al, Biotechnology 10:779-783 (1992)).

[0080] The term “immunoassay” is an assay that uses an antibody tospecifically bind an antigen, e.g., ELISA, western blot, RIA,immunoprecipitation and the like. The immunoassay is characterized bythe use of specific binding properties of a particular antibody toisolate, target, and/or quantify the antigen.

[0081] “Nucleic acid” refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

[0082] Unless otherwise indicated, a particular nucleic acid sequencealso implicitly encompasses conservatively modified variants thereof(e.g., degenerate codon substitutions) and complementary sequences, aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

[0083] The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

[0084] The term “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

[0085] Amino acids may be referred to herein by either their commonlyknown three letter symbols or by the one-letter symbols recommended bythe IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,likewise, may be referred to by their commonly accepted single-lettercodes.

[0086] “Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

[0087] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles of the invention.

[0088] The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M)

[0089] A “promoter” is defined as an array of nucleic acid controlsequences that direct transcription of a nucleic acid. As used herein, apromoter includes necessary nucleic acid sequences near the start siteof transcription, such as, in the case of a polymerase II type promoter,a TATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

[0090] The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

[0091] An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

[0092] In one embodiment of the invention the expression vector is aviral vector, preferably one that integrates into the host cell genome,such as a retroviral vector, or an adeno-associated viral vector.Examples of retroviruses, from which viral vectors of the invention canbe derived, include avian retroviruses such as avian erythroblastosisvirus (AMV), avian leukosis virus (ALV), avian myeloblastosis virus(ABV), avian sarcoma virus (ACV), spleen necrosis virus (SNV), and Roussarcoma virus (RSV); non-avian retroviruses such as bovine leukemiavirus (BLV); feline retroviruses such as feline leukemia virus (FeLV) orfeline sarcoma virus (FeSV); murine retroviruses such as murine leukemiavirus (MuLV), mouse mammary tumor virus (MMTV), murine sarcoma virus(MSV), and Moloney murine sarcoma virus (MoMSV); rat sarcoma virus(RaSV); and primate retroviruses such as human T-cell lymphotropicviruses 1 and 2 (HTLV-1, 2) and simian sarcoma virus (SSV). Many othersuitable retroviruses are know to those of skill in the art. Often theviruses are replication deficient, i.e., capable of integration into thehost genome but not capable of replication to provide infective virus.

[0093] In another embodiment of the invention, the vector is a transientvector such as an adenoviral vector, e.g., for transducing the cellswith a recombinase to delete the integrated oncogenes.

[0094] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

EXAMPLES

[0095] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of noncritical parameters that could be changed ormodified to yield essentially similar results.

Example I Preparation of Immortalized Human Pancreatic β-cell Lines

[0096] To develop cell lines from human β-cells, islet preparations wereenriched for β-cells based on flavin adenine dinucleotide (FAD)autofluorescence prior to retroviral infection, as described below (FIG.1). Immunohistochemical analysis showed that cells with low FADautofluorescence contained mostly α with a small number of β cells,while cells with high FAD autofluorescence (selected cells in box)contained 96% insulin-positive cells. Glucagon but not insulin wasdetectable by RIA in culture medium from the low FAD population, whileinsulin but not glucagon was detectable in culture medium from high FADcells.

[0097] The 96% pure insulin-positive population was used to create theβlox5 cell line by infection with the LTPRRNLlox retroviral vectorexpressing SV40 T antigen and H-ras^(val12) (Halvorsen et al., Molecularand Cellular Biology 19:1864-1870 (1999)). This cell line exhibits anextended lifespan and is immortalized by infection with a retroviralvector expressing the hTRT telomerase gene (Halvorsen et al., Molecularand Cellular Biology 19:1864-1870 (1999)). Insulin was detectable at lowlevels shortly after selection in G418, but was rapidly lost (FIG. 2A,lane 9). Furthermore, βlox5 does not express other pancreatic hormones(FIG. 2C).

[0098] To isolate the cells, a single cell suspension was prepared froman adult islet preparation of 80% purity. Islets with a diameter of50-100 μm were handpicked under direct vision using a stereoscope afterstaining with dethrone (60 μmol/L). Picked islets were incubatedovernight in RPMI containing 2.5 mM glucose, then dissociated intosingle cells using non-enzymatic dissociating solution (Sigma Corp., StLouis, Mo.) and shaking in a 37° C. water bath at 60 cycles/minute aspreviously described. The resulting single cell suspension was analyzedin a fluorescence-activated cell sorter (Facstar Plus, Becton Dickinson,Mansfield Mass.) and particle flavin adenine dinucleotide (FAD)autofluorescence (510-550 nm) was plotted against forward light scatter.Two islet cell populations became apparent when FAD autofluorescence(510-550 nm) was plotted against forward light scatter, as previouslydescribed for rat islets.

[0099] Highly autofluorescing cells were sorted into sterile Hanks BSS,washed and resuspended in RPMI containing 5 mM glucose and 10% FBS at aconcentration of 2×10⁶ cells/ml. Cells were aggregated into cellclusters by placing 50 μl droplets in a petri dish, gently inverting,and incubating overnight. Thereafter monolayers were initiated from thecell aggregates as previously described for adult islets on HTB-9 matrixin the presence of HGF/SF. After 2-3 days, when the cells were observedto be dividing, they were infected with high titer retroviral vector.

Example II Growth Factor Expression in βlox5 Cells

[0100] The expression of some of the transcription factors that areimportant in establishing and maintaining β-cell differentiated functionwas examined (FIG. 3). Most significantly, PDX-1, a homeodomaintranscription factor that is required for β-cell development andfunction, is not expressed in βlox5, as shown by RT-PCR (not shown) orEMSA (FIG. 3A, lane 2). This result differs from growth-stimulatedprimary cells, where PDX-1 continues to be expressed (Beattie et al.,Diabetes 48:1013-9 (1999)). BETA2, a bHLH factor important inneurogenesis as well as β-cells is not expressed in βlox5. CREB, whichplays a critical role in cAMP responsiveness in β-cells, is also notexpressed (FIG. 3D). However, Pax6, which is expressed in all endocrinecells, was expressed at high levels in βlox5, consistent with an originfrom primary endocrine cells. RIPE3b, a factor that binds to an elementin the insulin promoter but that has not yet been isolated, is presentas detected by EMSA (not shown). Overall, some, but not all, of thetranscription factors that play important roles in establishing andmaintaining β-cell function are expressed in βlox5 cells.

Example III Expression of Recombinant PDX-1 in βlox5 Cells

[0101] Previously, it has been shown that introduction of PDX-1 into acell line, TRM-6, derived from human fetal islets, resulted in anincrease in somatostatin gene expression. PDX-1 also actedsynergistically with cell-cell contact to further increase the level ofsomatostatin expression about 1,000-fold above the cells not expressingPDX-1, to a level similar to that found in normal human islets. Todetermine whether βlox5 could be induced to express pancreatic hormones,recombinant PDX-1 was expressed in βlox5 using a retroviral vector.While this resulted in functional PDX-1 expression in the cells (FIG.3A, lane 3), in contrast to TRM-6, βlox5 did not respond to PDX-1 andcell-cell contact with an increase in hormone expression (FIG. 2A, lane3, FIG. 2C, lane 3), despite the fact that PDX-1 expression in βlox5resulted in functional protein as evidenced by its ability to bind DNAin EMSA (FIG. 3A, lane 3). Therefore, other factors were examined fortheir ability to induce hormone expression in βlox5 cells expressingPDX-1. Activin and betacellulin have been reported to be able to induceendocrine differentiation in cell lines and primary cells (Mashima etal., Diabetes 48:304-9 (1999), Huotari et al., Endocrinology 139, 1494-9(1998), Mashima et al., J. Clin Invest 97:1647-54 (1996), Yamaoka etal., J Clin Invest 102:294-301 (1998)). However, they had no effect onhormone expression in βlox5 cells (data not shown).

Example III Treatment of PDX-1 Expressing Cells with GLP-1

[0102] GLP-1, a small peptide cleaved from the proglucagon molecule, isan insulin secretogogue Drucker et al., Diabetes 47:159-69 (1998)). Ahomolog of GLP-1 derived from Gila lizards, exendin-4, has recently beenfound to stimulate rodent β-cell differentiation in vitro and in vivo(Xu et al., Diabetes 48:2270-2276 (1999), Zhou et al., Diabetes48:2358-66 (1999). Therefore, exendin-4 was tested for its ability tostimulate β-cell differentiation in βlox5. 10 nmolar exendin-4 wasadministered to the cells for four days, and then removed from theculture medium.

[0103] By itself, exendin-4 had no effect on hormone expression in βlox5(FIG. 2A, lane 8). However, when βlox5 cells expressing PDX-1 were grownin suspension culture as three-dimensional aggregates in the presence ofthe exendin-4, a substantial amount of insulin mRNA was detectable (FIG.2A, lane 2). The nature of the interaction between exendin-4, cell-cellcontact, and PDX-1 is evident from the fact that no hormone mRNA wasdetectable with any one or two of the three inducing factors (FIG. 2A).Quantitative RT-PCR revealed that the level of insulin mRNA in inducedβlox5 cells was as that found in adult islets (FIG. 2B). Islet amyloidpolypeptide (IAPP) mRNA, a hormone that is co-secreted with insulin, wasalso expressed in induced βlox5 cells (FIG. 2C, lane 2). However, theother major pancreatic hormones, glucagon, somatostatin, and pancreaticpolypeptide (PP), were not induced (FIG. 2C).

Example IV Expression of Pancreatic Hormones in PDX-1 Expressing βlox5Cells

[0104] The expression of transcription factors important in β-celldevelopment was examined to determine whether they had been induced byPDX-1, cell-cell contact, and exendin-4. BETA2 was induced only by thecombination of all three inducing factors (FIG. 3C). Interestingly,CREB, which plays an important role in the cAMP response of β-cells, wasinduced by exendin-4 alone, independently of cell-cell contact and PDX-1(FIG. 3D). GLP-1 has been reported to increase cAMP levels and this hasbeen thought to be a major mechanism by which GLP-1 acts as an insulinsecretogogue (Serre et al., Endocrinology 139:4448-54 (1998)). This isthe first report that activation of the GLP-1 receptor can induce CREBexpression, suggesting that GLP-1 may act at multiple levels inpromoting cAMP signaling. RIPE3b, a factor that plays a critical role ininsulin gene expression but that has not yet been cloned, is expressedin βlox5 irrespective of PDX-1, cell-cell contact, or exendin-4 (FIG.3E).

Example V Glucose Responsiveness

[0105] A hallmark of functional β-cells is the ability to secreteinsulin in response to blood glucose. Primary β-cells store largequantities of insulin and secrete it in response to a variety ofphysiological stimuli, most significantly extracellular glucose. Inducedβlox5 cells have an insulin content of 6 picograms per microgram DNA at1.6 mM glucose. Intracellular insulin is easily detectable byimmunohistochemistry (FIG. 4A). Media from induced βlox5 cells containsmature insulin that comigrates with bovine insulin (FIG. 4B). A majorcomponent of the glucose-sensing apparatus is glucokinase (Matschinskyet al., Diabetes 47:307-315 (1998)). Induced βlox5 cells expressglucokinase mRNA, while uninduced cells do not (FIG. 5A). The extent towhich βlox5 cells exhibit glucose-responsive insulin secretion wastested by culturing βlox5 cells in different concentrations of glucoseand measuring insulin release in the culture medium by RIA (FIG. 5B).Half-maximal secretion occurred at approximately 11 mM, similar tonormal adult islets.

Example VI Transplantation of Glucose Responsive Cells in vivo

[0106] βlox5 cells expressing PDX-1 were transfected with an adenoviralvector expressing cre, to excise the recombinant SV40 T antigen andH-ras^(val12) genes, as described in Halvorsen et al., Molecular andCellular Biology 19:1864-1870 (1999). The cells had been previouslystimulated with exendin-4, as described above. The cells weretransplanted under the kidney capsule of nude mice for three months. Oneto ten million cells were transplanted in a mice that weighed about 25grams. After three months, the mice were bled for c-peptide and thegrafts were harvested. C-peptide was measured according to standardprocedures using RIA (FIG. 6). These cells exhibit glucose-responsiveinsulin secretion.

Example VII Expression of recombinant NeuroD/BETA2 and PDX-1 in βlox5Cells

[0107] It has been demonstrated that the expression of PDX-1 in βlox5cells, coupled with cell-cell contact and activation of GLP-1 receptorby treatment with its agonists, such as exendin-4, can induce the cellsto differentiate and become capable of glucose-sensitive insulinproduction (Dufayet de la Tour et al., Mole. Endocrinology15(3):476-483, 2001). To further determine the involvement ofNeuroD/BETA2 in this context, βlox5 cells expressing both NeuroD/BETA2and PDX-1 were established. Recombinant NeuroD/BETA2 and PDX-1 wereintroduced into βlox5 cells using two retroviral vectors. Clones werethen selected and the expression of both NeuroD/BETA2 and PDX-1 wasconfirmed. Insulin production by βlox5 cells expressing bothNeuroD/BETA2 and PDX-1 was detected by RT-PCR.

[0108] Cells were cultured in DMEM with 5.5 mM glucose and 10% FBS.Selection of clones was conducted in the same medium containing 300ug/ml Hygromycin B. Qualitative RT-PCR was performed according toItkin-Ansari et al., Mol. Endocrinol. 14:814-822 (2000).

Example VIII Induction of Insulin Production in βlox5 Cells Expressingboth NeuroD/BETA2 and PDX-1

[0109] In light of the previous finding that activation of GLP-1receptor is one of the necessary inducing factors for β-celldifferentiation, βlox5 cells expressing both NeuroD/BETA2 and PDX-1 werealso tested for insulin production, before and after stimulation byexendin-4. The cells were treated with 10 nM exendin-4 in the culturemedium for four days before the assay.

[0110] Similar to the results of the above-mentioned previous studies,exendin-4 treatment alone was again incapable of inducing insulinproduction in βlox5 cells expressing both NeuroD/BETA2 and PDX-1.Insulin production became clearly detectable upon exendin-4 treatment,however, when these NeuroD/BETA2 and PDX-1 expressing βlox5 cells weremaintained in suspension culture and formed three-dimentionalaggregates.

[0111] In order to compare the levels of insulin production induced byexendin-4 under the culture conditions permitting cell-cell contact,samples of βlox5 cells expressing only PDX-1 and βlox5 cells expressingboth NeuroD/BETA2 and PDX-1 were analyzed using quantitative RT-PCR.βlox5 cells expressing only PDX-1 and βlox5 cells expressing bothNeuroD/BETA2 and PDX-1 showed an approximately equal amount of inducedinsulin production, under very similar conditions of cell-cell contactand exendin-4 treatment. The insulin content and secretorycharacteristics of the βlox5/PDX-1/BETA2 cells are similar to those ofthe βlox5/PDX-1 cells, i.e. each has an insulin content about 10% thatof human islets and an EC50 of about 10 mM. In contrast to βlox5 cellsexpressing PDX-1 alone, cells expressing PDX-1 and NeuroD/BETA2 did notundergo cell growth arrest. Instead, PDX-1/NeuroD/BETA2 cells could begrown in culture for a substantial length of time and through multiplecell divisions.

[0112] Cell aggregation was induced as described by Itkin-Ansari et al,supra, 2000. Quantitative insulin RT-PCR was performed by interpolationfrom a standard curve constructed using a plasmid containing the humaninsulin cDNA, as described by Beattie et al., Diabetes, 48:1013-1019,1999.

Example IX Cell-Cell Contact Correlates with Down-Regulation of c-MycProtein Expression in βlox5 and TRM-6 Cell Lines

[0113] We have found that induction of δ-cell differentiation in thehuman cell line TRM-6 was associated with a decrease in the rate of cellproliferation. Both cell-cell contact and expression of the homeodomaintranscription PDX-1 factor resulted in decreased proliferation. However,the mechanism by which that inhibition of cell division occurs andwhether it is important in the process of differentiation are unknown.We examined the expression of c-Myc, which plays a role in cell cyclecontrol, in monolayer culture compared to three-dimensional cellaggregates. Western blot analysis of whole-cell extracts from monolayercultures and aggregates revealed greatly decreased c-Myc expression inboth TRM-6 and βlox5 cells expressing PDX-1 when grown as aggregatescompared to monolayer cultures.

[0114] For western blots, whole-cell extracts (10 μg of protein) frommonolayer cultures and aggregates of TRM-6 and βlox5 cells as well aswhole-cell extracts of the MycER expressing clones were used. Expressionof c-Myc and MycER was determined by Western blot analysis with thehuman c-Myc-specific monoclonal antibody Myc-9E10 (Santa CruzBiotechnology, Santa Cruz, Calif.) at a dilution of 1:250. The samemembrane was re-probed with anti-actin rabbit antibody A2066 (Sigma,Saint Louis, Mo.) in order to verify the protein equivalence whencomparing monolayers to aggregates. Western Blot for Phospho-CREB(Ser133) was performed using a polyclonal antibody (PhosphoPlusCREB(Ser133) Antibody Kit, Cell Signaling Technology, Beverly, Mass.) at adilution of 1:500. Bound antibody was detected with horseradishperoxidase-linked anti-mouse or anti-rabbit Ig (Amersham PharmaciaBiotech, Buckinghamshire, England) and ECL (Amersham Pharmacia Biotech,Buckinghamshire, England).

Example X Expression of MycER Fusion Protein in βlox5/PDX-1 andTRM-6/PDX-1 Cell Lines

[0115] To test the hypothesis that decreased c-Myc expression in cellaggregates plays a causal role in promoting differentiation by cell-cellcontact, we took advantage of the inducible properties of a fusionprotein between an estrogen receptor and c-Myc (Eilers, et al. Nature340:66-68 (1989)). MycER was expressed in TRM-6 and βlox5 by retroviralinfection followed by puromycin selection. Selected βlox5 and TRM-6cells expressed MycER as shown by western blot analysis. To determinewhether the fusion protein was active, the expression of cyclin E andcyclin A was determined since they are known downstream targets ofc-Myc. The addition of 4-OHTM to cells expressing the fusion protein ledto an increase in the expression of cyclin E mRNA in βlox5/PDX-1/MycERaggregates, as demonstrated by RT-PCR, but did not affect cyclin Aexpression.

Example XI c-Myc Induction Causes an Increase in Cell Proliferation

[0116] PDX-1 and cell-cell contact caused a decrease in cellproliferation in βlox5/PDX-1/MycER cells as shown by immunostaining forthe proliferation-associated nuclear antigen Ki-67. In monolayerculture, 77% of βlox5 cells expressed Ki67, while only 16% of the cellsin βlox5/PDX-1/MycER aggregates were Ki67 positive (n=300 cells in eachcase). Addition of 4-OHTM to the aggregates increased the percentage ofKi67 positive cells to 57%. In the absence of 4-OHTM, few cells in theaggregates were positive for Ki-67, while in the presence of 4-OHTM manycells throughout the aggregates were positive. This indicates that c-Mycis playing a causal role in cell contact induced growth arrest.

Example XII c-Myc Induction Ablates Pancreatic Hormone Expression

[0117] To determine whether the induction of differentiation in theTRM-6 and βlox5 cell lines requires downregulation of c-Myc expression,the effect of inducing c-Myc activity on hormone expression in cellsexpressing the MycER fusion protein was tested. 4-OHTM or the sameamount of ethanol carrier was added to TRM-6/PDX-1 and βlox5/PDX-1 cellsat the initiation of the four day period of aggregation required forhormone induction. In addition, 100 nM of the GLP-1 agonist exendin-4was added to βlox5/PDX-1, as activation of the GLP-1 receptor isnecessary for β-cell differentiation in that cell line (Itkin-Ansari, etal., Molec. Endocrinol. 14:814-822(2000)).

[0118] In the absence of 4-OHTM, TRM-6 aggregates expressed high levelsof somatostatin after four days of aggregation, while βlox5 aggregatesexpressed high levels of insulin. However, in the presence of 4-OHTM,neither somatostatin nor insulin mRNA was present in TRM-6 or βlox5,respectively. Insulin protein (273±70.5 pmoles/L) was detected by ELISAin supernatant from βlox5/PDX-1/MycER aggregates in the absence of4-OHTM but was undetectable in the presence of 4-OHTM. Data wereobtained from four independent experiments. To determine whether c-Mycactivation produced ablation of hormone expression as opposed to aswitch to a different endocrine lineage, the expression of the othermajor pancreatic hormones, somatostatin, glucagon and pancreaticpolypeptide, was measured in 4-OHTM-treated βlox5/PDX-1/MycER cells andfound to be absent.

Example XIII Inhibition of Differentiation by c-Myc is not Associatedwith Cell Death

[0119] While c-Myc is a potent stimulator of entry into the cell cycle,it can also act to induce apoptosis in vitro and in vivo. In atransgenic mouse expressing c-Myc under the control of the insulinpromoter, prolonged activation of c-Myc in β-cells led to extensive celldeath (Knaack, et al. Diabetes 43:1413-7 (1994)). Both of the cell linest studied express SV40 T antigen and so are disrupted in the p53 pathwaythat is required for apoptosis induction by c-Myc. Further, the level ofmRNA for the housekeeping gene porphobilinogen deaminase (PD) remainedconstant with c-Myc activation, arguing against the occurrence ofextensive cell death. However, to rule out the possibility thatoverexpression of c-Myc was leading to cell death, we assessed theviability of the aggregated cells in the presence and absence of 4-OHTM.Both βlox5/PDX-1/MycER control aggregates and the ones thatoverexpressed c-Myc were found to be viable. Some cells at the center ofthe control aggregates were dead as determined by uptake of ethidiumhomodimer-1 (red). We commonly observe this, particularly with largeaggregates, and attribute it to ischemic necrosis. The same analysis wasperformed on TRM-6/PDX-1/MycER aggregates with the same result.

Example XIV c-Myc does not Interfere with the Induction of TranscriptionFactors Involved in Insulin Expression

[0120] Induction of insulin gene expression in βlox5 cells is a complexprocess that involves changes in the pattern of expression of a largenumber of genes, particularly transcription factors involved inestablishment and maintenance of β-cell differentiation. To determinewhether the effect of c-Myc activation is global, i.e., inhibition ofthe entire β-cell differentiation program that is activated in βlox5, orrestricted to a subset of genes that includes the hormones, we studiedthe expression in βlox5 cells of transcription factors that areimportant in β-cell differentiation. By focusing on genes involved inthe control of insulin gene expression, we also hoped to gain insightsinto the mechanism by which c-Myc inhibits insulin gene expression.

[0121] βlox5 cells express Pax6 and RIPE3b in both the uninduced andinduced states. BETA2/NeuroD is present only when the cells are induced.The level of CREB protein increases dramatically with induction ofβ-cell differentiation. c-Myc activation did not result in a change inthe level of mRNA for Pax6 or with the induction of BETA2/NeuroD 1 geneexpression that occurs when βlox5 cells are induced to differentiate, asdetermined by RT-PCR. As shown by electrophoretic mobility shift assayanalysis (EMSA), although not quantitative, we found binding of PDX-1,BETA2/NeuroD1 or RIPE3b to the insulin promoter in the samples fromβlox5/PDX-1/MycER aggregates cultured with and without 4-OHTM. Moreover,Western blot demonstrated that CREB expression was not affected by c-Mycactivation. Therefore, the effects of c-Myc are selective for a subsetof genes in the β-cell.

[0122] EMSA for PDX-1 and RIPE3b were performed using probes derivedfrom the human insulin promoter A5 and C1 elements, respectively(Itkin-Ansari P, et al. Molec. Endocrinol. 14:814-822 (2000); German, etal. Diabetes 44:1002-1004 (1995)). EMSA for BETA2 was performed using aprobe derived from the human insulin promoter E2 Box5′cagcccccagccatctgccgacc3′.

Example XV c-Myc Activation Stimulates Translocation of PDX-1 from theNucleus to the Cytoplasm

[0123] PDX-1 plays a role in the establishment and maintenance of β-celldifferentiation. In both the βlox5 and TRM-6 cell lines, it is requiredfor the induction of hormone expression. Therefore, PDX-1 was a goodcandidate for being a downstream target for c-Myc in the inhibition ofhormone expression. Since, in the cell lines that we are studying, PDX-1is introduced exogenously using a retroviral vector and is transcribedfrom the retroviral LTR promoter, it was unlikely that c-Myc would beacting at the transcriptional level. Therefore, we investigated PDX-1cellular localization in βlox5/PDX-1/MycER cells in the presence andabsence of 4-OHTM. 4-OHTM-induced activation of c-Myc caused atranslocation of PDX-1 from the nucleus to the cytoplasm. However, manycells exposed to 4-OHTM continued to express PDX-1 in the nucleus andPDX-1 binding to a target DNA sequence could still be detected by EMSA.This could be due to the length of time that the cells were exposed to4-OHTM or to other pathways such as p38 (RK/SAPK2) that influences PDX-1localization and function.

[0124] Because PDX-1 localization has been reported to be influenced bythe p38 pathway, the activation state of p38 was studied using amonoclonal antibody specific for the phosphorylated/active form of theenzyme. In control and 4-OHTM-treated βlox5/PDX-1/MycER cells, activep38 was present in the cytoplasm. Therefore, c-Myc activation did notresult in any changes in p38 expression or activation, suggesting thatthe effects of c-Myc on PDX-1 localization may involve a differentpathway or may be downstream of p38.

Example XVI c-Myc Expression in Fetal and Adult Human Pancreatic Tissue

[0125] To extend the relevance of the results with the cell lines topancreatic growth and differentiation in vivo, we usedimmunohistochemistry to examine the expression of c-Myc in human fetaland adult pancreatic tissue. In fetal islet-like cell clusters, manycells stained positively for c-Myc, while in adult pancreas a muchsmaller number of positive cells were seen. Interestingly, in bothcases, the staining was predominantly cytoplasmic. Consistent with theresults from the cell lines, no cells that stained positively forhormones including insulin, somatostatin, glucagon or pancreaticpolypeptide-positive cells were positive for c-Myc. Double-staining forthe proliferation marker Ki-67 and c-Myc revealed that only a subset ofc-Myc-positive cells were positive for Ki-67. However, the greatmajority of Ki-67-positive cells had c-Myc staining, consistent with arequirement for c-Myc in the proliferation of pancreatic cells.

[0126] These studies show that c-Myc plays a central role in controllingthe inverse relationship between proliferation and differentiation inhuman pancreatic endocrine cells. In vivo, there are no examples ofpancreatic endocrine cells that continuously divide and express hormones(Schwitzgebel, et al. Development 127:3533-3542 (2000)). In vitro,stimulating primary human pancreatic endocrine cells to divide usinggrowth factors and extracellular matrix leads to a rapid decline inhormone expression, which can be partially recovered by aggregation ofthe cells into islet-like cell clusters in which cell division isinhibited.

[0127] This report is the first to demonstrate a causal relationshipbetween c-Myc expression and pancreatic endocrine cell differentiation.While the mechanism by which this occurs is complex, without intendingto limit the scope of the invention, it occurs at least in part byeffects on the subcellular localization of PDX-1. Initial results foundno detectable changes in the expression or function of the BETA2, Pax6,CREB, or RIPE3b transcription factors

[0128] In some settings, including the adult murine pancreas modeldescribed above (Pelengaris et al. Curr Opin Genet Dev 10:100-5 (2000)),deregulated c-Myc expression promotes apoptosis. In our system, noapoptosis was observed. It has been suggested that c-Myc mediatedapoptotic response depends on the p53 pathway via induction of ARF,which sequesters the p53 degrading protein, Mdm-2 (Sherr, et al. Curr.Opin. Genet. Devel. 10:94-9 (2000)). Our cell lines express SV40 large Tantigen, which interferes with both p16/Rb and p53/p21 pathways. Thecompromised p53 pathway may attenuate c-Myc apoptotic activity, therebyrevealing its effects on endocrine differentiation.

[0129] The current study is the first to directly demonstrate thatdownregulation of c-Myc is required for endocrine cell differentiationto proceed. Furthermore, the results suggest that there are at least twomechanisms involved in the control of endocrine cell division anddifferentiation, i.e., cell adhesion and PDX-1 function that convergeupon c-Myc. Cell-cell contact activates cell adhesion moleculesincluding cadherins that can downregulate c-Myc through the β-cateninpathway. E-cadherin has been shown to be important in pancreaticendocrine cell differentiation, raising the possibility that it isacting by downregulating c-Myc. Many genes that are repressed by c-Mycencode molecules involved in cell adhesion such as thrombospondin andintegrins αLβ2 (LFA-1) and α3β1, buttressing the role of thisproto-oncogene in the cell-cell contact triggered pathway.

[0130] The finding that c-Myc induces the translocation of PDX-1 to thecytoplasm provides important clues to the mechanism by which c-Myc mayact on pancreatic endocrine cell proliferation and differentiation. Inour system, not only does PDX-1 play a role in inducing hormoneexpression, but it also produces a marked growth arrest, particularly inthe β-cell line βlox5. The mechanism by which PDX-1 acts on the cellcycle is unknown, but the fact that c-Myc appears to inducetranslocation of PDX-1 to the cytoplasm indicates that c-Myc and PDX-1may act together in a pathway that controls both proliferation anddifferentiation. Continued administration of 4-OHTM to βlox5/PDX-1 cellsresults in rapid expansion of those cells, which ordinarily exhibitalmost complete growth arrest. Thus, cell-cell contact and PDX-1 eachplay dual roles in inhibiting entry into the cell cycle as well aspromoting differentiation.

[0131] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method for inducing insulin gene expression incultured endocrine pancreas β-cells, the method comprising the steps of:(i) expressing a recombinant NeuroD/BETA2 polynucleotide and arecombinant PDX-1 polynucleotide in endocrine pancreas β-cells that havebeen cultured under conditions such that the β-cells are in contact withother cells in the culture; and (ii) contacting the cells with a GLP-1receptor agonist, thereby inducing insulin gene expression in theβ-cells.
 2. The method of claim 1, wherein the GLP-1 receptor agonist isa GLP-1 analog.
 3. The method of claim 1, wherein the GLP-1 receptoragonist has an amino acid sequence of a naturally occurring peptide. 4.The method of claim 3, wherein the GLP-1 receptor agonist is GLP-1,exendin-3, or exendin-4.
 5. The method of claim 1, wherein the cells arecultured as aggregates in suspension.
 6. The method of claim 1, whereinthe β-cells are human β-cells.
 7. The method of claim 1, wherein theβ-cells express a recombinant oncogene.
 8. The method of claim 7,wherein the β-cells express more than one recombinant oncogene.
 9. Themethod of claim 1, wherein the β-cells express a recombinant telomerasegene.
 10. The method of claim 1, wherein the β-cells are βlox5 cells.11. A method of identifying a compound that modulates β-cell function,the method comprising the steps of contacting cells made by the methodof claim 1 with the compound and determining the effect of the compoundon β-cell function.
 12. A stable culture of endocrine pancreas β-cells,wherein the β-cells are in contact with other cells in the culture,wherein the β-cells express a recombinant PDX-1 polynucleotide and arecombinant NeuroD/BETA2 polynucleotide, and wherein insulin geneexpression is stimulated in the β-cells when exposed to an effectiveamount of a GLP-1 receptor agonist.
 13. The culture of claim 12, whereinthe GLP-1 receptor agonist is a GLP-1 analog.
 14. The culture of claim12, wherein the GLP-1 receptor agonist has an amino acid sequence of anaturally occurring peptide.
 15. The culture of claim 14, wherein theGLP-1 receptor agonist is GLP-1, exendin-3, or exendin-4.
 16. Theculture of claim 12, wherein the cells are cultured as aggregates insuspension.
 17. The culture of claim 12, wherein the β-cells are humanβ-cells.
 18. The culture of claim 12, wherein the β-cells express arecombinant oncogene.
 19. The culture of claim 18, wherein the β-cellsexpress more than one recombinant oncogene.
 20. The culture of claim 12,wherein the β-cells express a recombinant telomerase gene.
 21. Theculture of claim 12, wherein the β-cells are βlox5 cells.
 22. A methodof identifying a compound that modulates β-cell function, the methodcomprising the steps of contacting the culture of claim 12 with thecompound and determining the effect of the compound on β-cell function.23. A method of treating a diabetic subject by providing to the subjectcells that secrete insulin in response to glucose, the method comprisingthe step of administering to the subject an effective amount of cellsaccording to claim
 1. 24. A method of treating a diabetic subject byproviding to the subject cells that secrete insulin in response toglucose, the method comprising the steps of: (i) contacting a culture ofendocrine pancreas β-cells expressing a recombinant PDX-1 polynucleotideand a recombinant NeuroD/BETA2 polynucleotide with a GLP-1 receptoragonist, wherein the β-cells have been cultured under conditions suchthat the β-cells are in contact with other cells in the culture; and(ii) administering the β-cells to the subject, thereby providing to thesubject cells that secrete insulin in response to glucose.
 25. Themethod of claim 24, wherein the diabetic subject is a human.
 26. Themethod of claim 25, wherein the subject has Type I insulin dependentdiabetes.
 27. The method of claim 24, wherein the GLP-1 receptor agonistis a GLP-1 analog.
 28. The method of claim 24, wherein the GLP-1receptor agonist has an amino acid sequence of a naturally occurringpeptide.
 29. The method of claim 28, wherein the GLP-1 receptor agonistis GLP-1, exendin-3, or exendin-4.
 30. The method of claim 24, whereinthe β-cells are cultured as aggregates in suspension.
 31. An endocrinepancreas β-cell comprising a recombinant PDX-1 polynucleotide and arecombinant NeuroD/BETA2 polynucleotide.
 32. The β-cell of claim 31,wherein the β-cell is a human β-cell.
 33. The β-cell of claim 31,wherein the β-cell expresses a recombinant oncogene.
 34. The β-cell ofclaim 33, wherein the β-cell expresses more than one recombinantoncogene.
 35. The β-cell of claim 31, wherein the β-cell expresses arecombinant telomerase gene.